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Read splitting
Read-splitting is a default parameter in MinKNOW to split the informatic chimeras, as explained below. In our Kit 14 chemistry, we have observed increased follow-on rates, and with increased follow-on rates (the rate of the complement strand entering the pore directly after the template strand has passed through), we have observed a higher rate of concatemerisation compared to the Ligation Sequencing Kit (SQK-LSK110). We are classifying these reads as 'informatic chimeras' as they are not physically joined during the library preparation process.
With SQK-LSK110, we typically observe <2% concatemerisation and at this rate, it typically does not affect downstream applications. With, for example SQK-LSK112, we have observed a rate as high as 10%. Both MinKNOW (v21.11 and higher) and stand-alone Guppy (v5.1 and higher) now offer the option of splitting these reads.
As you can see from the following example, the majority of the informatic chimeras (yellow) are removed after splitting for a human (native) and E. coli (PCR) sample.
It is important to note that the read splitting function is not designed to split reads that are incorrectly ligated together during sample preparation. While these make up a small percentage of reads, users should take care with ligation steps to follow the protocol carefully to reduce the chance of creating them.
